Rickettsia conorii, the etiological agent of Mediterranean spotted fever (MSF), is transmitted to humans by tick bite and preferentially infects microvascular endothelium lining the vasculature leading to `rickettsial vasculitis'. R. conorii is closely related to R. rickettsii, which causes Rocky Mountain spotted fever prevalent in the Americas. An intriguing, but as yet completely unexplored, feature of the natural lifecycle of pathogenic rickettsiae is their ability to successfully transition between poikilothermic ticks and highly evolved mammals, suggesting spatiotemporal regulation of the transcriptome at various host interfaces. In spite of small genomes and the tendency for reductive evolution, the mechanism(s) through which rickettsiae adapt to varying host environments remain virtually unknown. Recently, small regulatory RNAs (srRNAs) have emerged as the most important post-transcriptional regulators in bacteria. Among these, trans-acting srRNAs implement their effects by directly binding to target mRNA(s) through an RNA chaperone, whereas cis-acting srRNAs are present on the anti-sense strand of an open reading frame. Together, these srRNAs regulate bacterial gene expression patterns via initiation/termination of transcription, stabilization/degradation of target mRNAs, and regulation of of translation. We have utilized a combination of bioinformatics tools to identify a total of 33 srRNAs (including 6S RNA, -tmRNA, and RNase P) in the R. conorii genome. We further demonstrate expression of at least 4 novel candidates, namely srRNA1-srRNA4, in both human microvascular endothelial cells and tick vector cells infected with R. conorii and intriguingly, differential expression of srRNA1 and (but not srRNA3 and 4) in human versus tick cells. These findings yield convincing foundation data in support of our hypothesis that srRNA-mediated regulation of transcriptome is an important determinant of rickettsial stress response, adaptation, and virulence depending on their intracellular host niches. Accordingly, Aim 1 proposes to identify all cis- and trans-acting srRNAs in R. conorii using differential RNA sequencing, followed by quantitative comparative analysis of the expression levels of novel rickettsial srRNAs in mammalian host (HMECs) and tick vector (RSE8) cells. Notably, an established RNA chaperone Hfq is absent in ~50% of bacteria, yet even those lacking Hfq have functional srRNAs and newly discovered RNA chaperones such as HP1334 in H. pylori. Rickettsiae lack hfq homologs in their genomes, implicating that as yet unidentified RNA chaperone(s) facilitate srRNA-mRNA interactions. Aim 2, therefore, proposes to identify and characterize an auxiliary RNA chaperone interacting with novel srRNA(s) and define its contributions to riboregulation in R. conorii. This proposal is highl relevant to the mission of the NIH, as it aims to identify novel small regulatory RNAs as well as a complementary chaperone in an NIAID category C priority pathogen during its interactions with the host (human) and vector (tick) cells by application of state-of-the-art approaches of genomics and proteomics.